The subject invention pertains to compounds which are useful as anti-inflammatory agents and to compositions containing such compounds as active ingredients. More particularly, the invention concerns novel biologically active bis-heterocyclic compounds, e.g. bis-indoles, novel uses of the compounds, pharmaceutical compositions containing these compounds, and methods of producing the compounds. The novel uses of the compounds relate to the anti-neurogenic inflammatory properties of the disclosed bis-heterocyclic compounds. Specifically exemplified herein are dragmacidin f, topsentin d and topsentin e, and their salts, analogs and derivatives.
The prevention and control of inflammation is often of great importance for the treatment of humans and animals. Much research has been devoted to development of compounds having anti-inflammatory properties. Certain methods and chemical compositions have been developed which aid in inhibiting or controlling inflammation, but additional anti-inflammatory methods and compositions are needed.
It has been found that some natural products and organisms are potential sources for chemical molecules having useful biological activity of great diversity. Marine sponges have proved to be such a source, and a number of publications have issued disclosing organic compounds derived from marine sponges. Such publications include Scheuer, P. J. Ed. (1978-1983) Marine Natural Products, Chemical and Biological Perspectives, Academic Press, New York; Faulkner, D. (1995) J. Nat. Prod. Rep. 12:223-269; (1994) 11:355-394; (1993) 10:497-539; (1992) 9:323-364; (1991) 8:97-147; (1990) 7:269-309; (1988) 5:613-663; (1987) 4:539-576; (1986) 3:1-33; (1984) 1:551-598.
Indole compounds of marine origin have also been described in Moquin, C., M. Guyot (1984) Tetrahedron Letters 25 (44):5047-5048; Norton, R. S., R. J. Wells (1982) J. Am. Chem. Soc. 104: 3628-3635; Roll, D. M., C. M. Iireland, H. S. M. Lu, J. Clardy (1988) J. Org. Chem, 53:3276; Kobayashi, H., T. Ohta, S. Nozoe (1990) Tetrahedron 46:7699; Jiminez, C., E. Quinoa, P. Crews, (1991) Tetrahedron Lett. 32:1843; Jiminez, C., E. QUinoa, M. Adamczeski, L. M. Hunter, P. Crews (1991) J. Org. Chem. 56:3403; Bifulco, G., I. Bruno, L. Minale, R. Riccio, A. Calignano, C. Debitus (1994) J. Nat. Prod. 57:1294; and Bifulco, G., I. Bruno, R. Riccio, J. Lavayre, G. Bourdy (1995) J. Nat. Prod. 58:1254.
Utilizing sponges as a source material and supplemented by synthetic production methods, new classes of biologically active compounds and new pharmaceutical compositions useful as antitumor and antiviral agents have been provided to the art. For example, bis-heterocyclic compounds such as bis-indoles have been previously described as having antimicrobial, antitumor or antiviral activity. Specifically, the bis-indole compounds known as topsentins are disclosed in U.S. Pat. No. 4,866,084. Dragmacidin and its related compounds isolated from the marine sponge of the Dragmacidon sp. are disclosed in U.S. Pat. No. 4,895,844. These patents are herein incorporated by reference. These compounds as well as the homocarbonyl topsentins and hamacanthins have also been described as having inhibitory activity against cellular inflammatory responses. See U.S. Pat. Nos. 5,290,777 and 5,464,835, which are also hereby incorporated by reference. The present invention provides additional related compounds which have improved water solubility characteristics and which have a novel utility as anti-neurogenic inflammatory compositions.
Other advantages and further scope of applicability of the present invention will become apparent from the detailed descriptions given herein; it should be understood, however, that the detailed descriptions, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent from such descriptions.
The objects of the present invention are accomplished by the provision of novel anti-inflammatory bis-heterocyclic compounds that have a general structure according to the formula:
A1xe2x80x94Mxe2x80x94A2 
wherein each of A1 and A2 is a heterocycle; and M is a core moiety linking the heterocycles, A1 and A2. In a specific embodiment, the compound comprises an indole as the A1 and A2 moieties. Thus, the compound can be a bis-indole. Other compounds of the subject invention can comprise heterocycles such as pyridyl or purine as the A1 and A2 moieties. The core moiety M can be a linear or cyclic group comprising at least three atoms.
Specifically exemplified herein are the novel compounds which have been designated as dragmacidin f, topsentin d, and topsentin e. Advantageously, these compounds have been found to possess anti-neurogenic inflammation activity.
As described herein, the invention also comprises pharmaceutical compositions, e.g. anti-neurogenic inflammatory compositions, containing as an active ingredient an effective amount, of one or more compounds according to the formula expressed above and a non-toxic, pharmaceutically acceptable carrier or diluent. The pharmaceutical compositions of the subject invention can further comprise other active compounds. Such other active compounds include, but are not limited to, anti-inflammatory compounds for example, steroidal compounds, including hydrocortisone and the like; or non-steroidal anti-inflammatories, including acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, indomethacin, and the like. The second active ingredient can include antiviral, antibacterial, antifungal or other antimicrobial compounds or antitumor compounds as well.
As described herein, the invention further comprises processes for the production of compounds and compositions of the invention and novel methods of use thereof, e.g. methods of inhibiting a neurogenic inflammatory response in a human or animal.
In accordance with the invention, methods for inhibiting inflammation comprise administering to a human or animal in need of such treatment an effective amount of the pharmaceutical composition
The subject invention pertains to novel uses as anti-inflammatory agents of bis-heterocyclic compounds and compositions comprising the bis-heterocyclic compounds. Surprisingly, the bis-heterocycle compounds of the subject invention can be highly effective in inhibiting neurogenic inflammation.
A specific embodiment of the subject invention is a bis-indole compound, wherein one of the indole rings has been reduced and an internal cyclization has taken place between a 2-aminoimidazolyl-ethylamine side chain and one of the indole rings as shown in structure (I) below: 
R1-4 are the same or different selected from xe2x80x94H, xe2x80x94OH, halogen, xe2x80x94R, xe2x80x94OR, xe2x80x94OCOR, xe2x80x94OA, or NZZ (wherein the Zs can be the same or different);
Y is the single group xe2x95x90O, or the single group xe2x95x90NZ, or two groups, same or different, selected from xe2x80x94H, xe2x80x94OH, xe2x80x94OR, xe2x80x94OCOR, NZZ (wherein the Zs can be the same or different);
Z is independently selected from xe2x80x94H, xe2x80x94R, xe2x80x94OH, or xe2x80x94COR;
R is C1-C8 alkyl or C1-C8 alkoxyl, mesyl, or tosyl; and A is xe2x80x94R-phenyl.
A particularly preferred embodiment is the compound called dragmacidin f (structure (II), below), and salts thereof. In this compound, R1xe2x95x90R2xe2x95x90R4xe2x95x90Zxe2x95x90H; R3xe2x95x90Br; Y is the single group xe2x95x90O. 
A specific embodiment of the invention includes compounds in which alkylation of the imidazole ring of the topsentins has occurred as shown in structure (III) below: 
wherein
R1-8 are the same or different selected from xe2x80x94H, xe2x80x94OH, halogen, xe2x80x94R, xe2x80x94OR, xe2x80x94OCOR, xe2x80x94OA, or NZZ (wherein the Zs can be the same or different);
R is C1-8 alkyl or aryl;
Z is independently selected from xe2x80x94H, xe2x80x94R, xe2x80x94OH, or xe2x80x94COR;
R is C1-8 alkyl or C1-8 alkoxyl, mesyl, or tosyl; and
A is xe2x80x94R-phenyl.
A particularly preferred embodiment is the compound in which R1xe2x95x90R2xe2x95x90R4xe2x95x90Zxe2x95x90H and R3xe2x95x90Br and R6xe2x95x90OH, and salts thereof, as shown below in topsentin d (IV): 
A specific embodiment of the invention includes compounds in which alkylation of the imidazole ring of the topsentins has occurred as shown in structure (V) below: 
wherein
R1-8 are the same or different selected from xe2x80x94H, xe2x80x94OH, halogen, xe2x80x94R, xe2x80x94OR, xe2x80x94OCOR, xe2x80x94OA, or NZZ (wherein the Zs can be the same or different);
R is C1-8 alkyl or aryl;
Z is independently selected from xe2x80x94H, xe2x80x94R, xe2x80x94OH, or xe2x80x94COR;
R is C1-8 alkyl or C1-8 alkoxyl, mesyl, or tosyl; and
A is xe2x80x94R-phenyl.
A particularly preferred embodiment is the compound in which R1xe2x95x90R2xe2x95x90R4xe2x95x90Zxe2x95x90H and R3xe2x95x90Br and R6xe2x95x90OH, and salts thereof, as shown below in topsentin e (VI): 
Skilled chemists having the benefit of the instant disclosure, can readily use procedures to prepare the subject compounds. In carrying out such operations, suitable filtration, chromatographic and other purification techniques can be used. These techniques could include, for example, reversed phase (RPLC), column, vacuum flash, medium pressure (MPLC) and high performance liquid chromatography (HPLC) with a suitable column such as silica gel, Sephadex LH-20, ammonia-treated silica gel, bonded phase RP-18, RP-8 and amino columns. Such columns are eluted with suitable solvents such as heptane, ethyl acetate, methylene chloride, methanol, isopropanol, acetonitrile water, trifluoroacetic acid (TFA) and various combinations thereof.
One method of preparation of the compounds used according to the subject invention involves extraction from marine sponges of the genus Spongosorites (Phylum Porifera, Class Demospongiae, Order Halichondrida, Family Halichondriidae). Certain of the samples used in connection with this invention have been assigned to the species Spongosorites ruetzleri (HBOM Catalog Numbers 003:00112, 003:00113, 003:00114, 003:00115, 003:00116, 003:00117, 003:00118, 003:00119, 003:00120, 003:00927); other specimens represent new species of Spongosorites (HBOM Catalog Numbers 003:00549 and 003:00696). For descriptions of these samples and other Spongosorites species, refer to Diaz., M. C., Pomponi, S. A. and Van Soest, R. W. M. (1993) xe2x80x9cA systematic revision of the central West Atlantic Halichondrida (Demospongiae, Porifera), Part III: Description of valid species,xe2x80x9d Scientia Marina, 57(4):283-306. All taxonomic voucher samples cited herein with HBOM Catalog Numbers are deposited in the Harbor Branch Oceanographic Museum, Fort Pierce, Fla. All voucher specimens are preserved in 70% ethanol with an expected shelf life of at least 30 years and are accessible to those skilled in the art for taxonomic identification purposes.
A novel use for the described compounds and compositions is their administration to an animal or human as an agent in the control of a neurogenic inflammatory response. Anti-inflammatory activity against cellular activation of specific immune cells, e.g., phorbol myristate acetate (PMA)-induced inflammation, has been described for the subject compounds. See U.S. Pat. Nos. 5,290,777 and 5,464,835, which are hereby incorporated by reference. However, it is well recognized that activity of a compound in inhibiting cellular activated inflammation (e.g., PMA-induced edema or inflammation) is not predictive or suggestive of that compound""s activity in inhibiting neurogenic inflammation, e.g., capsaicin-induced or resiniferatoxin (RTX)-induced edema or inflammation.
Therefore, the discovery that the subject compounds have inhibitory activity against neurogenic inflammation is unexpected and advantageous. Neurogenic inflammation is evoked by neuropeptides released from primary afferent nerve terminals and by other secondarily released inflammatory mediators. Specifically, neurogenic inflammation can be evoked by neuropeptides, such as substance P(SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and neurokinin A(NKA), released from primary afferent C-fiber nerve terminals and histamine, secondarily released from mast cells (Dray, A., [1992] xe2x80x9cNeuro pharmacological mechanisms of capsaicin and related substancesxe2x80x9d Biochem Pharm 44(4):611-15). For purposes of the subject invention, unless otherwise noted, the terms xe2x80x9cinflammationxe2x80x9d and xe2x80x9cinflammatory responsexe2x80x9d refer to any and all such neurogenic inflammatory reactions including, but not limited to, immune-related responses and/or allergic reactions to a physical, chemical, or biological stimulus. xe2x80x9cAnti-neurogenic inflammatory activity,xe2x80x9d as used herein, will be understood by those of ordinary skill in the art to mean biological activity inhibiting or controlling a neurogenic inflammatory response.
Anti-inflammatory activity can occur by modes of action which can include, but are not limited to, lipid-mediated inflammatory responses, e.g., (i) suppression of cellular activation of phospholipase A2, either directly (as is known for the anti-inflammatory compound, manoalide) or indirectly (as is known for the anti-inflammatory compound, hydrocortisone); (ii) by inhibiting, or controlling, cyclooxygenation of arachidonic acid, similar to the action of non-steroidal anti-inflammatory drugs; or (iii) by affecting lipooxygenase products of peroxidase reactions to arachidonic acid, or by non-lipid-mediated inflammatory responses, e.g., protease-induced inflammatory responses, and the like. In addition, it is known that capsaicin (CAP), the active constituent found in cayenne pepper, induces an acute neurogenic inflammatory response when applied topically to skin. CAP is a highly selective pain producing substance that selectively stimulates nociceptive and thermal-sensitive nerve endings in tissues by acting on a specific membrane receptor. The mode of action of capsaicin therefore differs significantly from phorbol myristate acetate (PMA)-induced inflammation. By comparison, PMA elicits its pro-inflammatory effects through cellular activation of specific immune cells, such as macrophages and neutrophils. Consequently, the pain response to PMA develops more slowly than the immediate, but transient, pain response to capsaicin.
The compounds and compositions of the subject invention advantageously can block the nociceptive (CAP-induced) inflammatory pathway, thereby providing a method for inhibiting neurogenic inflammation. Accordingly, the compounds can be used in the treatment of inflammation at sites where the primary activating factor is of neurogenic origin, e.g., inflammatory bowel disease or nepehritis. The subject compounds and compositions can be useful in the treatment of chronic pain, migraines, thermal-induced pain, such as sunburn, or other thermal and nociceptive pain, and chronic pain associated with arthritis. Uses can also include other inflammatory conditions that involve a neurogenic pain-producing component, e.g., certain metastic carcinomas or inflammation of the blood vessels.
The compounds of the subject invention can be used to treat a variety of skin conditions including, but not limited to, radiation irritation and burns (including UV and ionizing), chemical burns, rhinitis, thermal burns, reddening of the skin, and chemically indued lesions.
The compounds of the subject invention can also be used to treat allergic response and/or promote wound healing. This can include the use of the compounds in aerosol form for the treatment of acute allergic reactions such as acute asthmatic attack and in the treatment of inflammation of the lung caused by chemical exposure.
The compounds of the subject invention can also be used to treat systemic anaphylactic reactions in animals and man.
The compounds of the subject invention can also be used to treat conjunctivitis and inflammatory gum diseases.
Following are examples which illustrate procedures for practicing the invention. A more complete understanding of the invention can be obtained by reference to the following specific examples of compounds, compositions, and methods of the invention. It will be apparent to those skilled in the art that the examples involve use of materials and reagents that are commercially available from known sources, e.g., chemical supply houses, so no details are given respecting them. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.